Professor Susan Brooks

Abstract

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of ‘humanising’ proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems.

For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae.

Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins.

Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells.

In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

Lectins are naturally occurring carbohydrate-binding proteins that are ubiquitous in nature and highly selective for their, often incompletely characterised, binding partners. From their discovery in the late 1880s to the present day, they have provided a broad palette of versatile tools for exploring the glycosylation of cells and tissues and for uncovering the myriad functions of glycosylation in biological systems. The technique of lectin histochemistry, used to map the glycosylation of tissues, has been instrumental in revealing the changing profile of cellular glycosylation in development, health and disease. It has been especially enlightening in revealing fundamental alterations in cellular glycosylation that accompany cancer development and metastasis, and has facilitated the identification of glycosylated biomarkers that can predict prognosis and may have utility in development of early detection and screening, Moreover, it has led to insights into the functional role of glycosylation in healthy tissues and in the processes underlying disease. Recent advances in biotechnology mean that our understanding of the precise binding partners of lectins is improving and an ever-wider range of lectins are available, including recombinant human lectins and lectins with enhanced, engineered properties. Moreover, use of traditional histochemistry to support a broad range of cutting-edge technologies and the development of high throughout microarray platforms opens the way for ever more sophisticated mapping – and understanding – of the glycome.

The clinical outcome of radiation therapy is restricted due to acquired radio-resistance of a subpopulation of tumour cells that may cause tumour relapse and distant metastasis. While the effects of ionising radiation (IR) such as DNA damage and cell stress are well-documented, the potential role of IR in inducing invasive potential in cancer cells has not been broadly studied and aimed to be investigated in this study. MCF-7 cells irradiated with 0 Gy (control) or 2 Gy X-ray therapeutic dose of IR were assessed for cell viability, percentage of apoptotic cells, reactive oxygen species (ROS) levels, DNA fragmentation, Matrigel invasion, assessment of epithelial-mesenchymal transition (EMT) markers and Helix pomatia agglutinin (HPA) binding at 30 minutes, 4- or 24-hours post-IR. Reduction in cell viability, increase in apoptotic cells, ROS positive cells, and DNA fragmentation was observed, while functional invasiveness and EMT were exacerbated together with altered glycosylation in MCF-7 cells irradiated with 2 Gy X-ray compared to control cells. These findings indicate that despite the detrimental effects of 2 Gy X-ray IR on MCF-7 cells, a subpopulation of cells may have gained increased invasive potential. The exacerbated invasive potential may be attributed to enhanced EMT and altered glycosylation. Moreover, deregulation of Transforming growth factor beta (TGF-β) following IR may be one of the elements responsible for these changes, as it lies in the intersection of these invasion-promoting cell processes.The clinical outcome of radiation therapy is restricted due to acquired radio-resistance of a subpopulation of tumour cells that may cause tumour relapse and distant metastasis. While the effects of ionising radiation (IR) such as DNA damage and cell stress are well-documented, the potential role of IR in inducing invasive potential in cancer cells has not been broadly studied and aimed to be investigated in this study. MCF-7 cells irradiated with 0 Gy (control) or 2 Gy X-ray therapeutic dose of IR were assessed for cell viability, percentage of apoptotic cells, reactive oxygen species (ROS) levels, DNA fragmentation, Matrigel invasion, assessment of epithelial-mesenchymal transition (EMT) markers and Helix pomatia agglutinin (HPA) binding at 30 minutes, 4- or 24-hours post-IR. Reduction in cell viability, increase in apoptotic cells, ROS positive cells, and DNA fragmentation was observed, while functional invasiveness and EMT were exacerbated together with altered glycosylation in MCF-7 cells irradiated with 2 Gy X-ray compared to control cells. These findings indicate that despite the detrimental effects of 2 Gy X-ray IR on MCF-7 cells, a subpopulation of cells may have gained increased invasive potential. The exacerbated invasive potential may be attributed to enhanced EMT and altered glycosylation. Moreover, deregulation of Transforming growth factor beta (TGF-β) following IR may be one of the elements responsible for these changes, as it lies in the intersection of these invasion-promoting cell processes.

In humans, the UDP-N-α-D galactosamine:polypeptide N-acetylgalactosaminyltransferases family (ppGalNAc-Ts, GalNAc-Ts or GALNTs) comprises 20 isoenzymes. They are responsible for the initial synthesis of α-GalNAc1,3-O-Ser/Thr, or Tn antigen, at initiation of mucin type O-linked glycosylation. This structure is normally extended by the further sequential action of glycosytransferases to build more complex linear or branched O-linked structures, but in cancers it is frequently left unelaborated, and its presence is often associated with poor patient prognosis. Altered levels of GALNT expression or distribution have also been extensively reported in a wide range of cancers. These changes would be predicted to result in marked alterations in GalNAc O-linked glycosylation, including altered levels of site specific O-linked glycosylation and changes in the glycan structures formed, including, potentially, exposure of truncated O-glycans such as Tn antigen. Many reports have demonstrated that altered levels of specific GALNTs have prognostic significance in cancers, or shown that they are associated with changes in cell behaviour, including proliferation, migration, invasion or growth and metastasis in animal models.  We have previously reviewed how deregulation of GALNTs in several epithelial cancers is a feature of different stages metastasis. Here we consider evidence that changes in GALNT expression, and therefore consequent alterations in GalNAc O-linked glycosylation, may directly influence molecules implicated in aspects of epithelial-mesenchymal transition (EMT), a fundamental aspect of cancer metastasis, during which epithelial cancer cells lose their cell-cell junctions, apical-basal polarity and adhesive interactions with basement membrane and become mesenchymal, with a spindle-shaped morphology and increased migratory capacity.

To increase cancer patient survival and wellbeing, diagnostic assays need to be able to detect cases earlier, be applied more frequently, and preferably before symptoms develop. The expansion of blood biopsy technologies such as detection of circulating tumour cells and cell-free DNA has shown clinical promise for this. Extracellular vesicles released into the blood from tumour cells may offer a snapshot of the whole of the tumour. They represent a stable and multifaceted complex of a number of different types of molecules including DNA, RNA and protein. These represent biomarker targets that can be collected and analysed from blood samples, offering great potential for early diagnosis. In this review we discuss the benefits and challenges of the use of extracellular vesicles in this context and provide recommendations on where this developing field should focus their efforts to bring future success.

Along with the cells that are exposed to radiation, non-irradiated cells can unveil radiation effects as a result of intercellular communication, which are collectively defined as radiation induced bystander effects (RIBE). Exosome-mediated signalling is one of the core mechanisms responsible for multidirectional communication of tumor cells and their associated microenvironment, which may result in enhancement of malignant tumor phenotypes. Recent studies show that exosomes and exosome-mediated signalling also play a dynamic role in RIBE in cancer cell lines, many of which focused on altered exosome cargo or their effects on DNA damage. However, there is a lack of knowledge regarding how these changes in exosome cargo are reflected in other functional characteristics of cancer cells from the aspects of invasiveness and metastasis. Therefore, in the current study, we aimed to investigate exosome-mediated bystander effects of 2 Gy X-ray therapeutic dose of ionizing radiation on the invasive potential of MCF-7 breast cancer cells in vitro via assessing Matrigel invasion potential, EMT characteristics and the extent of glycosylation, as well as underlying plausible molecular mechanisms. The findings show that exosomes derived from irradiated MCF-7 cells enhance invasiveness of bystander MCF-7 cells, possibly through altered miRNA and protein content carried in exosomes.

While metastasis – the spread of cancer from the primary location to distant sites in the body – remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a ‘scratch’ or ‘wound-healing’ assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes.

Ionising radiation (IR) is commonly used for cancer therapy; however, its potential influence on the metastatic ability of surviving cancer cells exposed directly or indirectly to IR remains controversial. Metastasis is a multistep process, by which the cancer cells dissociate from the initial site, invade, travel through the blood stream or lymphatic system, and colonise distant sites. This complex process has been reported to require cancer cells to undergo epithelial-mesenchymal transition (EMT), by which the cancer cells convert from an adhesive, epithelial to motile, mesenchymal form, and is also associated with changes in glycosylation of cell surface proteins, which may be functionally involved in metastasis. In this paper, we give an overview of metastatic mechanisms and of the fundamentals of cancer-associated glycosylation changes. While not attempting a comprehensive review of this wide and fast moving field, we highlight some of the accumulating evidence from in vitro and in vivo models for increased metastatic potential in cancer cells that survive IR, focusing on angiogenesis, cancer cell motility, invasion and EMT and glycosylation. We also explore the indirect effects in cellsexposed to exosomes released from irradiated cells. The results of such studies need to be interpreted with caution and there remains limited evidence that radiotherapy enhances metastatic capacity of cancers in a clinical setting, and undoubtedly has a very positive clinical benefit. However, there is potential that this therapeutic benefit may ultimately be enhanced through a better understanding the direct and indirect effects of IR on cancer cell behaviour.

Ionising radiation (IR) is commonly used for cancer therapy; however, its potential influence on the metastatic ability of surviving cancer cells exposed directly or indirectly to IR remains controversial. Metastasis is a multistep process by which the cancer cells dissociate from the initial site, invade, travel through the blood stream or lymphatic system, and colonise distant sites. This complex process has been reported to require cancer cells to undergo epithelial-mesenchymal transition (EMT) by which the cancer cells convert from an adhesive, epithelial to motile, mesenchymal form and is also associated with changes in glycosylation of cell surface proteins, which may be functionally involved in metastasis. In this paper, we give an overview of metastatic mechanisms and of the fundamentals of cancer-associated glycosylation changes. While not attempting a comprehensive review of this wide and fast moving field, we highlight some of the accumulating evidence from in vitro and in vivo models for increased metastatic potential in cancer cells that survive IR, focusing on angiogenesis, cancer cell motility, invasion, and EMT and glycosylation. We also explore the indirect effects in cells exposed to exosomes released from irradiated cells. The results of such studies need to be interpreted with caution and there remains limited evidence that radiotherapy enhances the metastatic capacity of cancers in a clinical setting and undoubtedly has a very positive clinical benefit. However, there is potential that this therapeutic benefit may ultimately be enhanced through a better understanding of the direct and indirect effects of IR on cancer cell behaviour.

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumour growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so they represent an excellent potential source of novel biomarkers for the disease. In this review we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but a number of studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insight into the stage of the disease and predict the likely therapeutic outcome. There remain a number of challenges to the use of EVs as biomarkers, but through ongoing research and innovation in this exciting field there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome. 

Background. Aberrant glycosylation is a hallmark of cancer cells and plays an important role in oncogenesis and cancer progression including metastasis. This study aimed to assess alteration in cellular glycosylation, detected by lectin Helix pomatia agglutinin (HPA) binding, in adrenal cancers and to determine whether such altered glycosylation has prognostic significance. Methods. HPA binding lectin histochemistry was performed on archival paraffin wax‐embedded specimens of adrenocortical cancers excised from patients attending two tertiary referral centres. Benign tumours were used as controls. Demographic, histological and survival data were collected and compared between patients with HPA‐positive and HPA‐negative tumours. Results. Thirty‐two patients were treated for adrenal cancer between 2000 and 2016; their median age was 49 (range 23–79) years. Fifteen patients had functioning tumours (14 adrenal Cushing's tumours and 1 Conn's tumour). Mean(s.d.) tumour size was 127·71(49·70) mm. None of 10 control tumours expressed HPA‐binding glycoproteins. Invasion was associated with HPA‐binding glycoproteins (P = 0·018). Local recurrence or metastatic disease did not significantly differ between HPA‐positive and HPA‐negative adrenocortical cancers. Overall survival was significantly longer in patients with HPA‐negative tumours (median survival not reached versus 22 months in patients with HPA‐positive tumours; P = 0·002). Conclusion. Altered cellular glycosylation detected by lectin HPA is associated with poor survival in patients with adrenocortical cancer.

Pubmed, Web of Science and EBSCOhost were searched using keywords and synonyms for molecular, metastasis, breast cancer and bone to identify articles published between January 2004 and August 2016. 4,491 potentially relevant citations were retrieved. 63 articles met the inclusion criteria, which were primary studies reporting evidence of molecular factors that have functional roles in predisposing breast cancer bone metastasis in vivo.12 of those 63 articles that additionally met quality criteria were included in the review. Extracted data were tabulated and key findings that indicated biological mechanisms involved in breast cancer metastasis to bone were synthesised. 

15 proteins expressed by breast cancer cells were identified as factors that mediate breast

cancer bone metastasis: ICAM-1, cadherin-11, osteoactivin, bone sialoprotein, CCN3, IL-11,

CCL2, CITED2, CXCR4, CTGF, OPN, CX3CR1, TWIST1, adrenomedullin and Enpp1.

Upregulation or overexpression of one or more of them by breast cancer cells resulted in

increased breast cancer metastasis to bone in vivo, except for CCL2 where bone-metastatic

cells showed a reduced expression of this factor. All factors identified, here expressed by

breast cancer cells, are proteins that are normally expressed in the bone microenvironment

and linked to physiologic bone functions. All have a functional role in one of more of the

following: cell proliferation and differentiation, bone mineralization and remodeling, cell

adhesion and/or chemokine signaling. Six of them (cadherin-11, ICAM-1, OPN, CX3CR1,

CCN3 and osteoactivin) have a reported function in cell adhesion and another eight (CCN3,

osteoactivin, Enpp1, IL-11, CTGF, TWIST1, adrenomedullin and CITED2) are reported to be

involved in cell proliferation and differentiation.
 

This review collates and synthesises published evidence to increase our understanding of

the biology of breast cancer osteomimicry in the development of bone metastasis. Findings

of this review suggest that changes in expression of proteins in breast cancer cells that

confer ostemimicry facilitate homing to bone to enable the development of bone metastasis. 

Ovarian cancers have a high mortality rate; this is in part due to resistance to the platinum-based compounds used in chemotherapy. In this paper, we assess the role of microRNA-31 in the development of chemoresistance to cisplatin. We used previous data from microarray experiments to identify potential microRNAs (miRNAs) involved in chemoresistance. The functional significance of these microRNAs was tested using miRNA mimics. We used RNA-seq to identify pathways and genes de-regulated in the resistant cell line and then determined their role using RNAi. Analysis of publically available datasets reveals the potential clinical significance. Our data show that miR-31 is increased, whilst potassium channel calcium activated large conductance subfamily M alpha, member 1 (KCNMA1), a subunit of calcium-regulated big potassium (BK) channels, is reduced in resistant ovarian cells. Over-expression of miR-31 increased resistance, as did knockdown of KCNMA1 or inhibition of BK channels. This suggests that these genes directly modulate cisplatin response. Our data also suggest that miR-31 represses KCNMA1 expression. Comparing the levels of miR-31 and KCNMA1 to cisplatin resistance in the NCI60 panel or chemoresistance in cohorts of ovarian cancer tumours reveals correlations that support a role for these genes in vitro and in vivo. Here we show that miR-31 and KCNMA1 are involved in mediating cisplatin resistance in ovarian cancer. Our data gives a new insight into the potential mechanisms to therapeutically target in cisplatin resistance common to ovarian cancer.

OBJECTIVE:

Ovarian cancer is the deadliest gynaecological cancer. A major contributor to the poor survival rate is the development of chemoresistance to platinum-based therapies such as cisplatin and carboplatin. Here we aimed to test the role of miRNAs in the acquisition of drug resistance in ovarian cancer.

METHODS:

We used microarrays to measure miRNA levels in the ovarian cancer cell line A2780 and its cisplatin-resistant derivative CP70. The role of miRNAs and the mRNA targets were tested using transfected miRNA mimics and siRNAs, respectively. Potential in vivo significance was investigated by analysing RNA levels in cohorts of ovarian cancer patients.

RESULTS:

We identified several miRNAs that are increased in cisplatin-resistant cells. We show that most of these do not directly contribute to cisplatin resistance. Interestingly, miR-21-3p, the passenger strand of the known oncomiR, directed increased resistance to cisplatin in a range of ovarian cell lines. This effect was specific to the star strand, as miR-21-5p had the opposite effect and actually increased sensitivity of A2780 cells to cisplatin. We identify NAV3 as a potential target of miR-21-3p and show that knockdown of NAV3 increases resistance. Exosomes released by CP70 cells were also capable of increasing resistance in A2780 cells, although this was independent of miR-21-3p. Finally, we use publically available transcriptomic data to demonstrate that miR-21-3p is raised, while NAV3 is reduced, in ovarian tumours that are resistant to platinum treatment.

CONCLUSION:

Our data suggest that miR-21-3p can induce cisplatin resistance in ovarian tumours, potentially by targeting the NAV3 gene.

Immunocytochemistry, the identification of cell- or tissue-bound antigens in situ, by means of a specific antibody-antigen reaction, tagged microscopically by a visible label, has a remarkably wide range of applications. The basic techniques are straightforward and can be adapted to explore the localisation of virtually any molecule of interest to the researcher in samples of normal and/or malignant cells. Heterogeneity can be mapped and loss or gain of immunoreactivity with tumour progression can be visualised. In this chapter, methodologies are given for appropriate preparation of cells and tissues, including cells cultured on coverslips (which can be used for live cell imaging), cell smears, frozen (cryostat) and fixed, paraffin wax-embedded tissue sections. Heat- and enzyme-based antigen retrieval methods are covered. Basic detection methods, which can be readily adapted, are given for direct (labelled primary antibody), simple indirect (labelled secondary antibody), avidin-biotin (biotinylated primary antibody), avidin-biotin complex (ABC), peroxidase-anti-peroxidase or alkaline phosphatase-anti-alkaline phosphatase (PAP or APAAP), and polymer- based methods. The use of enzyme labels including horseradish peroxidase and alkaline phosphatase, and fluorescent labels, are considered.

Lectins are naturally occurring carbohydrate-binding molecules. A very wide range of purified lectins are commercially available which exhibit a diversity of carbohydrate-binding preferences. They can be used in the laboratory to detect carbohydrate structures on, or in, cells and tissues in much the same way that purified antibodies can be employed to detect cell- or tissue-bound antigens using immunocytochemistry. As lectins can distinguish subtle alterations in cellular glycosylation, they are helpful in exploring the glycosylation changes that attend both transformation to malignancy and tumour progression. In this chapter, methodologies are given for appropriate preparation of many types of cell and tissue preparations, including cells cultured on coverslips (which can be used for live-cell imaging), cell smears, and frozen (cryostat) and fixed, paraffin wax-embedded tissue sections. Heat- and enzyme-based carbohydrate retrieval methods are covered. Basic detection methods, which can be readily adapted to the researcher's needs, are given for direct (labelled lectin), simple indirect (labelled secondary antibody directed against the lectin), and avidin-biotin (biotinylated lectin) and avidin-biotin complex. The use of both the enzyme label, horseradish peroxidase, and fluorescent labels is considered.

Binding of the lectin Helix pomatia agglutinin (HPA) has been shown to be associated with poor prognosis in many human cancers, but not in thyroid cancer. The aims of the present study were to assess alteration in cellular glycosylation, detected by HPA binding, in thyroid tumors; to determine if such altered glycosylation carries any prognostic significance; and to analyze qualitative and quantitative differences of HPA-binding glycoproteins in various thyroid tumors. Methods Lectin histochemistry was performed on 110 archival paraffin wax embedded specimens of various thyroid tumors excised between the years 1983-1993. Demographic data, histological data, and time to death were recorded, and multivariate Cox regression analysis was performed to determine a prognostic model for patient survival based on the data. Helix pomatia agglutinin binding glycoproteins were isolated from 128 fresh specimens of various thyroid tissues by affinity chromatography, analyzed by SDS-PAGE and western blotting. Results There was a marked qualitative difference in the profile of HPA-binding glycoproteins, with the malignant thyroid tumors showing a heterogeneous profile of numerous HPA binding glycoprotein bands. Lectin histochemistry showed significant positive HPA binding in malignant thyroid tumors (p = 0.0009). Kaplan-Meir survival analysis revealed that among patients who died of their disease those with HPA-positive tumors had a significantly shorter survival (p = 0.009). Conclusions This is the first study showing that HPA binding glycoproteins are synthesized by thyroid tumors. Thyroid cancers show a broader profile of HPA binding glycoproteins compared to benign thyroid tumors, and positive HPA binding is significantly associated with shorter survival and poorer prognosis in thyroid cancers.

Introduction: Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Materials and Methods: Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Results: Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. Conclusions: This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

Metastasis, the process by which cancer cells leave the primary tumour, disseminate and form secondary tumours at anatomically distant sites, is a serious clinical problem as it is disseminated disease, which is often impossible to eradicate successfully, that causes the death of most cancer patients. Metastasis results from a complex molecular cascade comprising many steps, all of which are interconnected through a series of adhesive interactions and invasive processes as well as responses to chemotactic stimuli. In spite of its clinical significance, it remains incompletely understood. This review provides an overview of some of the molecular interactions that are critical to metastasis. It summarises the principle molecular players in the major steps of the metastatic cascade. These are: (1) tumour angiogenesis, (2) disaggregation of tumour cells from the primary tumour mass, mediated by cadherins and catenins, (3) invasion of, and migration through, the basement membrane (BM) and extracellular matrix (ECM) surrounding the tumour epithelium, and subsequent invasion of the BM of the endothelium of local blood vessels. This is mediated through integrins and proteases, including urokinase form of plasminogen activator (uPA), matrix metalloproteinases (MMPs) and cathepsins, (4) intravasation of the tumour cells into the blood vessels prior to hematogeneous dissemination to distant sites, (5) adhesion of the circulating tumour cells to the endothelial cell lining at the capillary bed of the target organ site. This occurs through adhesive interactions between cancer cells and endothelial cells involving selectins, integrins and members of the immunoglobulin superfamily (IgSF), (6) invasion of the tumour cells through the endothelial cell, layer and surrounding BM (extravasation) and target organ tissue and (7) the development of secondary tumour foci at the target organ site.

Thyroid cancers are the most common endocrine malignancy. Radiation exposure, family history of thyroid cancer and some inherited conditions are the most important predisposing factors for the development of thyroid cancer. Three mitogenic signalling pathways have been described in the thyroid cell, which are influenced by various stimulatory and inhibitory hormones, growth factors and neurotransmitters. Various proto-oncogenes and oncogenes like ras, braf, trk, met and RET also play a role in the signal transduction systems. Two theories have been described in thyroid cancer pathogenesis, the foetal cell carcinogenesis theory and the more common, multistep carcinogenesis theory. The multistep carcinogenesis theory is now the accepted model in many human cancers, including thyroid cancer. The early events of tumour formation are the consequence of activation of either various growth factors or the proto-oncogenes like ras, met or ret. This results in the formation of differentiated thyroid cancers like the papillary, follicular or Hurthle cell cancers. The later stages of tumour formation involve further activation of proto-oncogenes and loss or inactivation of tumour suppressor genes like p53. Based on this theory, follicular carcinomas are generated from follicular adenomas and papillary carcinomas from precursor cells generated from thyrocytes. Anaplastic carcinoma may develop from papillary or follicular carcinoma by dedifferentiation. In this review article, we highlight the molecular pathogenesis of thyroid tumours.

More than half of human proteins are glycosylated by a bewildering array of complex and heterogeneous N- and O-linked glycans. They function in myriad biological processes, including cell adhesion and signalling and influence the physical characteristics, stability, function, activity and immunogenicity of soluble glycoproteins. A single protein may be glycosylated differently to yield heterogenous glycoforms. Glycosylation analysis is of increasing interest in biomedical and biological research, the pharmaceutical and healthcare industry and biotechnology. This is because it is increasingly apparent that glycosylation changes in diseases, such as cancer, making it a promising target for development of clinically useful biomarkers and therapeutics. Furthermore, as the non-human cells employed in expression systems glycosylate their proteins very differently to human cells, and as glycosylation changes unpredictably under changing environmental conditions, glycans analysis for quality control, optimum efficacy and safety of recombinant glycoproteins destined for human therapeutic use is paramount. The complexities of carbohydrate chemistry make analysis challenging and while there are a variety of robust methodologies available for glycan analysis, there is currently a pressing need for the development of new, streamlined, high throughput approaches accessible to non-specialist laboratories.

More than half of human proteins are glycosylated by a bewildering array of complex and heterogeneous N- and O-linked glycans. They function in myriad biological processes, including cell adhesion and signalling and influence the physical characteristics, stability, function, activity and immunogenicity of soluble glycoproteins. A single protein may be glycosylated differently to yield heterogenous glycoforms. Glycosylation analysis is of increasing interest in biomedical and biological research, the pharmaceutical and healthcare industry and biotechnology. This is because it is increasingly apparent that glycosylation changes in diseases, such as cancer, making it a promising target for development of clinically useful biomarkers and therapeutics. Furthermore, as the non-human cells employed in expression systems glycosylate their proteins very differently to human cells, and as glycosylation changes unpredictably under changing environmental conditions, glycans analysis for quality control, optimum efficacy and safety of recombinant glycoproteins destined for human therapeutic use is paramount. The complexities of carbohydrate chemistry make analysis challenging and while there are a variety of robust methodologies available for glycan analysis, there is currently a pressing need for the development of new, streamlined, high throughput approaches accessible to non-specialist laboratories.

Whilst there are many common features in the glycosylation pathways of all eukaryotic cells, the cells of different species often glycosylate their proteins very differently. This is of particular interest when the cells of non-human species, such as bacteria, plants, yeast or non-human mammalian cells, are used to express glycoproteins for potential human therapeutic use, as these proteins may be glycosylated with glycans very different to those typical of human cells. In this presentation the similarities and differences between human cell glycoprotein glycosylation and the glycosylation of other types of cells will be discussed briefly and some interesting – and biologically important – features of glycosylation highlighted. Some of the advances and challenges in biotechnology and engineering glycosylation pathways in non-human cells types will be described.

Aberrant glycosylation is an established characteristic of cancer cells, and appears to have a functional role in metastasis – the process by which cancer cells spread around the body. The lectin from Helix pomatia, the Roman snail (Helix pomatia agglutinin, HPA) recognises cancer-associated aberrant glycans terminating in the monosaccharide α-N-acetylgalactosamine (GalNAc). The presence of these glycans, detected by HPA binding, is associated with metastasis and consequent poor survival. We are investigating the putative functional role of these glycans in cancer cell adhesion to, and migration through, the endothelium (lining blood vessels) during metastasis. Eight breast cell lines, characterised for synthesis of GalNAc-glycans of interest, have been employed in investigations; HMT 3522, is derived from normal breast, and stably synthesises negligible levels of GalNAc-glycans, and the other cell lines; BT474, MDA MB 435, MDA MB 468, ZR751, MCF7, T47D and DU4475, are derived from breast cancer and stably synthesise increasingly greater amounts of GalNAc-glycans, consistent with their increasing metastatic ability.

A rocking adhesion assay was used to investigate the adhesion of the breast cell lines to an endothelial cell monolayer derived from human brain microvasculature, a site physiologically relevant to breast cancer metastasis. The effect of masking the GalNAc-glycans with HPA was investigated. The results from these assays provide novel evidence that GalNAc-glycans have a functional role in cancer cell adhesion to endothelial cells. It was also found that the HPA binding profiles of the breast cell lines appear to correlate with their abilities to adhere to endothelial cells via GalNAc-glycans.

Six human breast cancer cell lines were injected subcutaneously into scid mice and their in vivo growth behaviour and HPA binding pattern were analysed. Furthermore, the role of HPA binding glycoconjugates concerning the adhesion to endothelial cells in vitro was investigated.

Four of the tested cell lines engrafted in the scid mouse model but they showed considerable variations concerning their growth behaviour, their metastatic potential and their HPA binding pattern. HPA inhibited adhesive interactions between cell lines derived from metatstatic sources and tumour necrosis factor (TNF)α stimulated endothelial cells.

The transplantation of HPA defined breast cancer cell lines into scid mice is a useful animal model for the research of breast cancer and its metastasis. The HPA binding glycoconjugates appear to be associated with adhesive interactions between metastasising tumour cells and endothelial cells.

The prognosis for the majority of patients suffering from a solid neoplasm remains bleak, due to the fact that most cancers have already formed metastases at distant sites. Any progress in cancer therapy will, therefore, depend on the understanding of the metastatic cascade and therapies derived from this understanding. The development of clinically relevant models of metastases, using lectin-defined human cancer cell lines and severe combined immunodeficient (SCID) mice, is described. In conjunction with modern imaging techniques, these models will help to elucidate the molecular mechanisms governing the metastatic spread of tumours.

One of the commonest and least well understood posttranslational modifications of proteins is their glycosylation. Human glycoproteins are glycosylated with a bewilderingly heterogeneous array of complex N- and O-linked glycans, which are the product of the coordinated activity of enzymes resident in the endoplasmic reticulum and Golgi apparatus of the cell. Glycosylation of proteins is highly regulated and changes during differentiation, development, under different physiological—and cell culture—conditions and in disease. The glycosylation of recombinant proteins, especially those destined for potential administration to human subjects, is of critical importance. Glycosylation profoundly affects biological activity, function, clearance from circulation, and crucially, antigenicity. The cells of nonhuman species do not glycosylate their proteins in the same way as human cells do. In many cases, the differences are profound. Overall, the species most distant to humans in evolutionary terms, such as bacteria, yeasts, fungi, insects and plants—the species used most commonly in expression systems—have glycosylation repertoires least like our own. This review gives a brief overview of human N- and O-linked protein glycosylation, summarizes what is known of the glycosylation potential of the cells of nonhuman species, and presents the implications for the biotechnology industry.

Helix pomatia lectin (HPA) binding is a marker of metastatic competence in several human cancers. The altered cellular glycosylation detected by HPA is of clinical interest and functional significance, but research has been hampered by uncertainty over appropriate and accessible histochemical methods. Most studies have employed a complex multi-layered detection system localising binding of unconjugated HPA by layering with a polyclonal antibody to HPA, a biotinylated secondary antibody against the first antibody and streptavidin peroxidase. This detection system is sensitive and yields accurate prognostic information, but is lengthy and requires antibodies against HPA that are not widely available. A simpler technique, that uses peroxidase-labelled HPA is inappropriate as the carbohydrate-combining characteristics of the lectin are altered, and the prognostic significance of lectin binding is lost. Therefore a valid alternative, simple and accessible technique is required. In the present study, we compare the results of labelling of HPA binding using the complex multi-layered detection system with a simple avidin-biotin method. In a series of 101 breast cancers, both methods gave comparable results. Therefore, the avidin-biotin method appears to be appropriate for studies on HPA binding to detect altered glycoforms in cancer. It is hoped that its adoption may encourage research into this clinically significant alteration in cellular glycosylation.

The expression of the aberrant N-acetylgalactosamine (GalNAc) glycoconjugates, detected by binding of the lectin from Helix pomatia (HPA) is reported to be associated with metastatic competence and poor prognosis in a range of human adenocarcinomas, but the functional significance of the glycoconjugates in metastatic mechanisms is unknown. We have employed seven cell lines derived from normal breast epithelium, primary breast cancer and breast cancer metastases which stably express varying levels of HPA-binding glycoconjugates consistent with their derivation and phenotype. These cell lines have been thoroughly characterised and express identical profiles of HPA-binding glycoconjugates as tumour cells derived from clinical samples. Their ability to adhere to, and invade through, basement membrane components was investigated in a matrigel assay system, and the functional role of the aberrant GalNAc glycans assessed by competitive inhibition experiments using HPA. The behaviour of the cell lines in these assay systems was entirely consistent with their derivation and phenotype, but there was no evidence that the glycoconjugates of interest were functionally involved in adhesion or invasion mechanisms. Research in our laboratory is ongoing to seek a functional role for the HPA-binding glycoconjugates in other aspects of the metastatic cascade.

Over-expression of N-acetylgalactosamine glycoproteins as detected by binding of the lectin from Helix pomatia (HPA), is associated with metastatic competence and poor patient prognosis in a range of human adenocarcinomas. These glycoproteins remain poorly characterised, and their functional role has yet to be elucidated. This study describes characterisation of a range of human breast/breast cancer cell lines for the expression of the N-acetylgalactosaminylated glycoproteins of interest, and their comparison with normal breast epithelium and a range of clinical breast carcinoma samples. Confocal and light microscopy studies revealed cytochemical HPA-binding patterns consistent with a fundamental disruption in normal glycobiosynthetic pathways attending increasing metastatic potential. We report the most complete comparative analysis of HPA-binding ligands from cultured breast cells, clinical breast carcinoma samples and normal breast epithelium to date. Lectin blotting identified 11 major HPA-binding glycoprotein bands common to both clinical tumour samples and breast cell lines and 6 of these bands were also expressed by samples of normal breast epithelium, albeit at much lower levels. Moreover, very marked quantitative but not qualitative differences in levels of expression consistent with metastatic capability were noted.

Predicting long-term outcome after breast-cancer diagnosis remains problematic, particularly for patients with clinically small, axillary lymph node- negative tumours. Evidence suggests that the lectin Helix pomatia agglutinin (HPA) identifies oligosaccharides associated with poor-prognosis cancer. Our aim was to identify oligosaccharides that bind HPA in aggressive breast cancers. Breast-cancer cell lines (MCF-7, BT-549 and BT-20) and a cell line from human milk (HBL-100), which showed a range of HPA-binding intensities, were used to extract HPA-binding glycoproteins. Oligosaccharides were released using anhydrous hydrazine and separated on a range of HPLC matrices. We investigated whether HPA-binding oligosaccharides from cell lines were present in human breast-cancer tissues, using 69 breast-cancer specimens from patients with between 5 and 10 years' follow-up. A monosialylated oligosaccharide was over-expressed in the cell line that bound HPA strongly. Further analysis by normal-phase HPLC showed that the 2-aminobenzamide-conjugated oligosaccharide had a hydrodynamic volume of 4.58 glucose units (HPAgly1). Increased expression of HPAgly1 was associated with HPA staining of breast-cancer specimens (Student's t-test p = 0.025). Analysis of oligosaccharide levels and disease-free survival after treatment for breast cancer indicated a shorter disease-free interval for patients with elevated levels of HPAgly1. This is the first time that histochemical lectin staining has been correlated with biochemical mapping of oligosaccharides. Using this approach, we have identified a monosialylated HPA lectin-binding oligosaccharide present in breast-cancer cells grown in vitro which is elevated in breast-cancer specimens that bind the lectin.

Binding of the lectin from Helix pomatia (HPA), which recognises N-acetylgalactosamine and N-acetylglucosamine glycans, is a predictor of metastasis and poor prognosis in a number of human adenocarcinomas, including breast cancer. The glycoproteins to which it binds in these tumours have been only partially characterised, and the mechanisms underlying their biosynthesis remain unknown. In this study, 111 primary breast cancers were assessed for binding of HPA and labelling characteristics were compared directly with those of Dolichos biflorus agglutinin and soybean agglutinin, both of which also recognise N-acetylgalactosamine, Griffonia simplicifolia agglutinin II, which recognises N-acetylglucosamine, and Limax flavus agglutinin, Sambucus nigra agglutinin and Maackia amurensis lectin I, all of which recognise sialic acids. Results indicate that the HPA-binding partners expressed by cancer cells are predominantly N-acetylgalactosamine glycans, but some recognition of N-acetylglucosamine species is also likely. There was no evidence to support the hypothesis that overexpression of these moieties results from failure in sialylation. Alternative mechanisms, for example alterations in levels of activity of appropriate glycosyl transferases or disruption in transport and processing mechanisms leading to failure of normal chain extension of glycans may be responsible, and these are areas that warrant further investigation.

The lectin from Helix pomatia, the Roman snail (HPA), recognises terminal alpha N-acetylgalactosamine residues. A large number of lectin histochemical studies have demonstrated that expression of HPA-binding glycoproteins by cancer cells to be a marker of metastatic competence and poor prognosis in a range of common human adenocarcinomas, including those of breast, stomach, ovary, oesophagus, colorectum, thyroid and prostate. Around 80% of metastases arising from primary breast cancer are predictably HPA positive, but, intriguingly, around 20% do not express HPA binding glycoproteins reflecting the complexity of metastatic mechanisms and the further disruptions in cellular glycosylation that attend tumour progression. HPA binding is not an independent prognostic factor, but is strongly associated with the presence of metastases in local lymph nodes. It does appear to be independent of other clinical features of prognostic importance such as tumour size, histological grade, S-phase fraction, ploidy, and there is little convincing evidence of any association with oncogene expression or hormone receptor positivity. The precise nature of the metastasis-associated HPA binding partner(s) is a question of some interest, but thus far remains unclear. HPA will recognise, for example, the Tn epitope and blood group A antigen, but its prognostic significance appears to be through recognition of a much broader and heterogeneous array of N-galactosaminylated glycoproteins. Their synthesis appears to be mediated through alteration in expression or activity of one or more of the enzymes of glycosylation. The most likely putative roles of HPA-binding ligands in the metastatic cascade may be enhancement of invasive capacity, or interaction with an as yet unidentified lectin-like receptor facilitating adhesion processes. The prognostic information provided by HPA lectin histochemistry may be used clinically to inform the physician and aid treatment decisions; far more interesting is the challenge of further understanding the precise nature of the HPA-binding ligands, and defining their role in the complex mechanisms of metastasis.